Cultivation and Genetic Manipulation of Free-Living and Pathogenic Leptospires

自由生活和致病性钩端螺旋体的培养和基因操作

• 批准号: 8946511
• 负责人: PATRICIA A ROSA
• 金额: $ 10.82万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8946511
• 关键词: Acetylation; Affect; Area; Bacteria; Borrelia burgdorferi; Cell membrane; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Collection; Culture Media; DNA; Data; Development; Disease; Fluorescent Dyes; Frequencies; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Techniques; Genome; Goals; Growth; Homologous Gene; In Vitro; Infection; Intention; Knowledge; Laboratories; Length; Leptospira; Leptospirosis; Life; Lyme Disease; Maps; Measures; Membrane Proteins; Microbial Genetics; Modeling; Mutagenesis; National Institute of Allergy and Infectious Disease; Operon; Order Spirochaetales; Organism; Pathogenicity; Phenotype; Phosphorylation; Physiological; Physiology; Plasmids; Play; Post-Translational Protein Processing; Proteins; Proteomics; Publishing; Refractory; Relative (related person); Reporting; Research; Role; Shuttle Vectors; Source; System; Techniques; Technology; Testing; Time; Transcript; Virulence Factors; Virus Diseases; Work; antibody conjugate; density; genetic manipulation; improved; member; mutant; neglect; pathogen; promoter; recombinase; tool; vaccine development

Leptospirosis is a global, zoonotic disease caused by members of the genus Leptospira. Although widespread and sometimes fatal, leptospirosis is considered a neglected and understudied disease. The causative agent of Leptospirosis was first identified in 1916 but the slow in vitro growth rate and limited genetic tools with which to manipulate the genome of this spirochete have hampered the identification of virulence factors and development of a vaccine. Leptospires can be broadly divided into two groups: free-living saprophytes and infectious pathogens. The most widely used and studied species are L. biflexa (a non-pathogenic saprophyte) and L. interrogans (a pathogen). However, the non-pathogenic L. biflexa is more easily cultivated and more amenable to genetic manipulation than the pathogenic L. interrogans. Therefore, we have focused on L. biflexa to master the microbial and genetic techniques needed to manipulate this genus, with the intention to transfer this expertise to the more refractory pathogenic strains. Targeted gene inactivation, shuttle vector transformation, and transposon mutagenesis have all been successfully used in L. biflexa. To date, no shuttle vector system exists for pathogenic species and there are few published reports of targeted gene inactivation in L. interrogans. Transposon mutagenesis can be applied to L. interrogans but it functions at such a low efficiency that it cannot be utilized for any broad applications, such as auxotrophic screens or signature tagged mutagenesis. The lack of a shuttle vector for L. interrogans hinders complementation and thus limits interpretation of any resulting phenotypes of transposon or targeted deletion mutants. Since L. biflexa has a better transformation frequency than other species we plan to optimize new techniques in this organism. In FY2014 we have begun to evaluate different systems that may affect the transformation effiencies of leptospires. The lamda red recombinase system has been used successfully in other bacteria to improve targeted mutagenesis. We have begun to assess this system in L. biflexa, and if it appears promising, we will test it in the pathogen L. interrogans. Also, we are studying the CRISPR/Cas system that is present in L. interrogans but absent in L. biflexa. This system targets and degrades foreign DNA and we hypothesize that it may contribute to the lower transformation frequency observed in the pathogen relative to the saprophyte. Specifically, we have demonstrated that the CRISPR/cas operon is transcribed during in vitro growth and have integrated part of the operon into L. biflexa and have shown that the genes are also transcribed in this heterologous host. Currently, we are attempting to inactivate specific cas genes in L. interrogans and move the entire operon into L. biflexa. We proceeded in FY2014 to develop a proteomic map of in vitro cultivated L. biflexa to identify highly expressed proteins from membrane- and soluble-fractions. We have identified abundantly-expressed proteins that can be used as cellular markers, as controls for gene expression studies, and also quantified the transcript data from a subset of these genes. Further, we demonstrated that a significant number of L. biflexa proteins are subject to post-translational modification including phosphorylation and acetylation. Highly expressed proteins allow us to identify targets that may play important physiological roles and also use as tagged proteins for various expression studies. This work is being completed with an internal collaboration with Dr. James Carroll in the Laboratory of Persistent Viral Diseases, NIAID and Dr. Lisa Olano of the Research Technologies Branch, NIAID. The long-term objective of this project is to use the improved tools and techniques to understand the basic physiology of leptospires and the mechanisms of infection and pathogenecity of L. interrogans. Together this knowledge should help accelerate the development of preventative measures against Leptospirosis.

钩端螺旋体病是一种由钩端螺旋体属成员引起的全球性人畜共患疾病。尽管钩端螺旋体病广泛存在，有时甚至致命，但它被认为是一种被忽视和研究不足的疾病。钩端螺旋体病的病原体于 1916 年首次被识别，但该螺旋体的体外生长速度缓慢，且用于操纵该螺旋体基因组的遗传工具有限，阻碍了毒力因子的鉴定和疫苗的开发。 钩端螺旋体可大致分为两类：自由生活的腐生菌和传染性病原体。使用和研究最广泛的物种是 L. biflexa（一种非致病性腐生菌）和 L. interrogans（一种病原体）。然而，非致病性双折乳杆菌比致病性询问乳杆菌更容易培养，也更容易进行基因操作。因此，我们将重点放在双弯乳杆菌上，以掌握操纵该属所需的微生物和遗传技术，旨在将这种专业知识转移到更难治的致病菌株上。靶向基因失活、穿梭载体转化和转座子诱变均已成功应用于 L. biflexa。迄今为止，尚不存在针对致病物种的穿梭载体系统，并且很少有关于问号钩体中靶向基因失活的报道。转座子诱变可应用于问号钩端螺旋体，但其作用效率如此之低，以至于不能用于任何广泛的应用，例如营养缺陷型筛选或特征标记诱变。问号钩端螺旋体穿梭载体的缺乏阻碍了互补，从而限制了对转座子或靶向缺失突变体的任何结果表型的解释。由于 L. biflexa 比其他物种具有更好的转化频率，我们计划优化该生物体的新技术。 2014财年我们开始评估可能影响钩端螺旋体转化效率的不同系统。拉姆达红重组酶系统已成功用于其他细菌，以改善定向诱变。我们已经开始在双折乳杆菌中评估这个系统，如果它看起来有希望，我们将在病原体问号乳杆菌中测试它。此外，我们正在研究问号钩体中存在但双折钩体中不存在的 CRISPR/Cas 系统。该系统靶向并降解外源 DNA，我们假设它可能导致病原体相对于腐生菌的转化频率较低。具体来说，我们已经证明 CRISPR/cas 操纵子在体外生长过程中转录，并将部分操纵子整合到 L. biflexa 中，并表明这些基因也在该异源宿主中转录。目前，我们正在尝试灭活问号钩体中的特定 cas 基因，并将整个操纵子转移到双折钩体中。 我们在 2014 财年着手开发体外培养的 L. biflexa 的蛋白质组图谱，以鉴定膜组分和可溶组分中高表达的蛋白质。我们已经鉴定出丰富表达的蛋白质，可以用作细胞标记、基因表达研究的对照，并且还量化了这些基因子集的转录数据。此外，我们证明大量双曲乳杆菌蛋白受到翻译后修饰，包括磷酸化和乙酰化。高表达的蛋白质使我们能够识别可能发挥重要生理作用的靶标，并可用作各种表达研究的标记蛋白质。这项工作是在与 NIAID 持续性病毒性疾病实验室的 James Carroll 博士和 NIAID 研究技术部门的 Lisa Olano 博士的内部合作下完成的。 该项目的长期目标是利用改进的工具和技术来了解钩端螺旋体的基本生理学以及询问钩端螺旋体的感染和致病机制。这些知识共同有助于加速钩端螺旋体病预防措施的制定。